Coupon reader

ABSTRACT

A method of reading a coupon channel that displays a test section pattern after being exposed to a target substance, the method uses a device having a computer readable memory, digital camera, logic assembly and user interface; providing a pixel target intensity profile; placing the coupon in the device and exposing the coupon channel to a test fluid mixture; automatically using the digital camera to take a digital image of the coupon channel test section after the exposure. The improvement in the method includes finding the contiguous set of pixels from the test section of the coupon channel that best matches the intensity profile of the target pattern representation and determining if this best match set of pixels exceeds a similarity threshold and in response to a best match set of pixels passing the similarity threshold, automatically providing a human perceptible indication that the target substance has been detected.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 15/487,397 filed Apr. 13, 2017, which claims the benefit ofU.S. Provisional Application No. 62/322,357, filed Apr. 14, 2016, whichare both incorporated by reference as if fully set forth herein.

FIELD OF THE INVENTION

The embodiments herein relate to devices and methods for reading acoupon that displays a pattern when a target substance is detected.

BACKGROUND

A “coupon reader” is a device that accepts a “coupon,” typically aplanar tray having a chemically reactive section that is imbued with achemical mixture that is designed to react to a target substance andforms a visually detectable indicator pattern after a predetermineddevelopment time. A common implementation is the lateral flowimmunoassay. The term “ticket” is also sometime used to refer to acoupon. Generally, coupons are designed to be read by a humantechnician, although coupon readers are also becoming common. The readerautomates detection of the indicator pattern, removing reliance on humaninterpretation. Typically, a single company makes both coupons and thereaders for those coupons, but there are also companies that do not makecoupons, but do make coupon readers for coupons made by other companies(“third-party coupon readers”). Some coupons have only one section thatreacts to a single target substance, whereas other coupons have a numberof sections, each one designed to react to a different target substance.Coupons come in various sizes, but it appears that no currentlyavailable coupons are greater than 15 cm in any dimension.

Coupons are used to detect substances of interest in the medical andpublic safety fields. For example, some coupons are designed to accept abodily fluid onto the chemically reactive sections, to detect anaturally occurring target compound, such as a hormone, or a toxin. Apublic safety coupon can accept samples from many sources, includingaerosolized particulates, liquids, and solids. Air-derived samples,mixed and concentrated into a liquid solution, and placed onto thechemically reactive sections, can provide an indication of an aerosolpoison. Typically, the sample is incorporated into a buffer solutionthat is applied to the coupon, to facilitate the exposure of the couponchemicals to the atmospheric agent or test specimen. Each chemicallyreactive section is referred to as a channel. Coupons may have multiplechannels, each detecting a different target substance, allowing formultiple assays on a single coupon. Each channel typically includes acontrol section, which will develop in tandem with the test section ofthe coupon (the portion of the coupon on which the test line willappear), but unlike the test section will display the indicator pattern,whether or not the target substance is present. The control sectionperforms two functions, first, if the control section does not displaythe indicator pattern it is generally an indication that something hasgone wrong with the process of exposure. Accordingly, a negative readingdoes not, in that instance, indicate an absence of the target substance,but only indicates a test that was not performed correctly. Also, itshows the test personnel what the indicator pattern looks like.

In many situations, it is important to obtain a quick result from thecoupon exposure, but the coupon manufacturer may have designated a setperiod of time for the indicator pattern to develop. Human techniciansmay set the coupon aside and attend to other tasks while the coupondevelops for the manufacturer-specified time period. Variation in buffersolution and how it is applied can cause the same coupon to developdifferently even when exposed to the same concentration of targetsubstance. The use of a reader can lead to a faster detection of theindicator pattern when compared to a human, particularly in low light orstressful situations.

For the purpose of automatically analyzing the visually detectableindicator pattern of the coupon using a camera, it is desirable toprovide uniform illumination over the surface of the coupon, as thisallows full use of the camera's dynamic range over the entire field ofview. As an example, if pixels directly under the light source have areflected intensity of 255 units while more dimly illuminated pixels atthe edge of the coupon have a maximum reflected intensity of 128, thenthe dynamic range and resolution of a measurement may be lower by afactor of two for assay channels near the coupon edge as compared tochannels directly under the light source. In addition, if light raysilluminate a particular part of the coupon surface at oblique anglesonly, shadows can be created that confound analysis of assay results.These issues are compounded by the fact that a compact portable deviceis desirable, as it is more easily moved from place to place. Mostbioassay coupons are on the order of 11 cm in maximum diagonalmeasurement, and in the interests of having a compact device, it isdesirable that the distance from the coupon to the camera lens is ofsimilar magnitude. To maximize the number of pixels in the analysis, itis also essential that the coupon fill the field of view as much aspossible. That is, a near-field macro imaging design is preferred.

Light sources typically used for camera systems are so-called “whiteLEDs.” Uniform near-field illumination is sometimes accomplished using acircular array of white LEDs surrounding the camera lens, referred to asa ring light. Ring lights may have multiple circular rings of LEDs andmay contain more than 100 LEDs. Such a large number of LEDs couldsignificantly reduce battery life and thus operation lifetime in abattery-operated instrument. In addition, commercially-available ringlights still produce an illumination field in which intensity peaks inthe center and drops off radially, albeit much more gradually than witha single LED. Since bioassay coupons are typically rectangular, ringlights may not provide uniform edge-to-edge illumination.

Using a single LED light source is far preferable, but the hotspotproblem must be solved. The LED cannot be mounted on the same axis asthe camera lens: It must be physically located to the side of the lens,and the hotspot consequently appears at an off-axis camera image point.This does not improve the situation since parts of the coupon remainfarther away than others from the LED, and light intensity over thecoupon surface can be significantly nonuniform across the face of thecoupon.

Some improvement in light uniformity can be realized if a piece ofground glass or opalescent glass is mounted directly under the lightsource. This causes light to be re-emitted in a Lambertian pattern byvirtue of the ground glass's surface roughness or by internal refractionin the opalescent glass. That is, the distribution of light emergingfrom the surface approximates a cosine function relative to a surfacenormal. These methods more broadly disperse the light, but there stillremains a radial distribution of ever-decreasing light intensityrelative to an axis passing through the LED source's center point andperpendicular to its surface.

Therefore, there remains a need in the art for improved methods ofreading a coupon that, among other things, minimize or compensate forvariations in illumination over the surface of the coupon.

SUMMARY

The following embodiments and aspects thereof are described andillustrated in conjunction with systems, tools and methods which aremeant to be exemplary and illustrative, not limiting in scope. Invarious embodiments, one or more of the above-described problems havebeen reduced or eliminated, while other embodiments are directed toother improvements.

In a first separate aspect, the present invention may take the form of amethod of reading a coupon channel that displays a test pattern in atest section after being exposed to a target substance, the methodincludes providing a device having a computer readable memory, a digitalcamera, a logic assembly and a user interface; providing a color-ratiopixel target pattern representation, representative of an exposed and atleast partially developed coupon channel control section pattern;placing the color-ratio pixel target pattern into the memory; placingthe coupon in the device and exposing the coupon channel to a test fluidmixture; automatically using the digital camera to take a digital imageof the coupon channel test section after the exposure, the digital imageformed of color pixels, each containing at least two sub-pixelsrepresenting the intensity of two different colors. The improvement inthe method includes for each color pixel forming a color-ratio pixel byforming the ratio of a first one of the two subpixels with the other,and automatically using the logic assembly to compare the color-ratiopixels in the test section as shown in the digital image to thecolor-ratio pixel target pattern representation to determine if the testsection passes a similarity threshold; and in response to a test sectionpassing the similarity threshold, automatically providing a humanperceptible indication that the target substance has been detected.

In a second separate aspect, the present invention may take the form ofa method of reading a coupon channel that displays a test sectionpattern after being exposed to a target substance, the method uses adevice having a computer readable memory, digital camera, logic assemblyand user interface; providing a pixel target intensity profile; placingthe coupon in the device and exposing the coupon channel to a test fluidmixture; automatically using the digital camera to take a digital imageof the coupon channel test section after the exposure. The improvementin the method includes finding the contiguous set of pixels from thetest section of the coupon channel that best matches the intensityprofile of the target pattern representation and determining if thisbest match set of pixels exceeds a similarity threshold and in responseto a best match set of pixels passing the similarity threshold,automatically providing a human perceptible indication that the targetsubstance has been detected.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thedrawings and by study of the following detailed descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced drawings. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1 is a block diagram of a preferred embodiment.

FIG. 2 is an illustration of common exposed coupon channel combinationsof test and control pattern indications.

FIG. 3 is a cross section of an apparatus for reading coupons accordingto one preferred embodiment, showing the position of the coupon relativeto the apparatus and the path taken by light passing through thenegative axicon lens.

FIG. 4 is a graph showing the elimination of light intensity effects ina developed coupon pattern due to defining a ratiometric signalvariable.

FIG. 5 is a graph showing the application of least-squares nonlinearcorrelation to identify line strength and pixel location in a developedcoupon pattern.

DETAILED DESCRIPTION

Referring to FIG. 1, which is a block diagram of a preferred embodiment,in one preferred embodiment a coupon reader 10 includes a generallytight light chamber (see FIG. 3, as defined by walls 38) which isadapted to permit the introduction of any currently commerciallyavailable coupon (anything that is smaller than a 15 cm×10 cm×1 cmcuboid). The reader 10 includes a user interface 12 that prompts a userto enter the type of coupon being introduced into reader 10. In oneembodiment interface 12 includes a liquid crystal display coupled with afew keys (not shown), for making a choice of options presented on thedisplay, and for providing a user perceptible indication. Interface 12may also include a light or sound indicator for providing the humanperceptible indication. In another embodiment, a voice recognitionelement (not shown) is included. In yet another embodiment, a USB port(not shown) is provided for a keyboard or a computer to be connected forcommands and information to be sent to reader 10. A computer readablememory 14 and a microcontroller 24, driven by a clock 22, collectivelyforms a logic assembly for controlling a digital camera 16 andinterpreting digital imagery taken by camera 16, to form a detection, ordetermine that no detection has occurred. A GPS receiver 20 determineslocation, which is input to microcontroller 24. The location informationtogether with test results is sent by way of a cellular networktransceiver 18 over a cellular network link 28 to a remote station 30,for storage. In one preferred embodiment, remote station 30 includes amapping feature permitting users to see test results displayed on a mapat the locations where the various test results were formed.

In one embodiment, means are provided to introduce a target patternrepresentation (an image of a developed coupon) into a computer readablememory 14 of reader 10. This data entry may be performed by placing adeveloped coupon or a control section into reader 10 and using userinterface 12 to command reader 10 to use a digital camera 16 to take adigital photograph of the developed coupon and store it in the computerreadable memory 14, properly labeled as a digitized image of a targetpattern. In another preferred embodiment, reader 10 is provided withdigitized target images already stored. Otherwise digitized targetimages may be introduced into reader 10 by way of the USB port (notshown) or may be downloaded by way of a cellular network transceiver 18.

After an exposed coupon is introduced into reader 10, themicrocontroller 24 controls the digital camera 16 to repeatedly formimages of each coupon channel and effectively compare each image withthe digitized target image stored in computer readable memory 14.

Coupon manufacturers typically identify a specific time period thatshould elapse between exposing the coupon to the substance to be testedand reading the results on the coupon. In some cases, results may bevisible before this predetermined development time has elapsed and timemay be wasted by continuing to wait for the entire pre-determined timeperiod. The ability of the coupon reader 10 to determine when anindicator pattern has been detected and to alert any nearby people thatdetection has occurred helps to eliminate this wasted time. It will beappreciated that this ability is particularly important when the couponreader is being used to detect the presence of toxic or harmfulcompounds in a public safety context, where time may be of the essence.

Many other algorithms, including least squares detection and variouslinear algorithms are used in alternative embodiments. FIG. 2 is anillustration of test patterns that are commonly encountered and whichare amenable to interpretation and quantification using the mathematicaland algorithmic approaches revealed herein. The left half of the couponshown would typically be referred to as the “control section” and theright half as the “test-section” where the test line appears.

Prior to channel analysis, all embodiments require that the coupon imageis recognized in the digital image being analyzed. Immediately followingcoupon image capture, image recognition routines analyze the capturedimage and identify channel locations based on the detection ofcharacteristic coupon features. In embodiments, the user has entered thecoupon type, and this information facilitates the recognition of thechannel locations. The processing described below relates to eachchannel analyzed.

Known optical features of the coupon are pre-loaded into the devicebefore use. Image recognition tools identify the coordinates of thesecharacteristic features for each different coupon type. The pixelcoordinates of these features provide reference points and allow eachcoupon image to be overlaid by a coordinate map which can then be usedto locate the assay channels.

Upon positive recognition and satisfaction of location criteria, couponanalysis is allowed to continue. Upon detection of an abnormalsituation, such as the coupon being inserted 90 or 180 degrees from thecorrect orientation, the user is alerted to the type of error. Once theerror is remedied and location criteria are satisfied, sample analysisprocedures may proceed.

In one embodiment, each time a reader 10 processes a coupon, the resultsare time, position and device code stamped, with position being providedby a GPS reader 20, time being provided by a clock 22, and device beingprovided by device firmware, all controlled by microcontroller 24 andstored in computer readable memory 14. In a preferred embodiment, thisinformation is uploaded periodically via cell network link 28 using cellnetwork transceiver 18, to a remote station 30. In one embodiment,remote station 30 includes a display and may be commanded to display amap of an area, showing the locations, times, results and device codesof each coupon reading.

Referring to FIG. 2, a coupon channel is shown at different end resultsof exposure. illustration 1 shows a coupon channel in which neither testline T nor control line C developed. This could indicate an unusedcoupon channel but could also indicate a coupon channel that has notbeen properly exposed to buffer solution. The absence of a controlpattern indication, in this instance, prevents a false conclusion thatthe target substance is not present. Illustration 2 is a weak positiveindication, due to the weak pattern. Illustration 3 is a strongpositive, whereas illustration 4 shows an absence of the targetsubstance. Illustration 5 indicates that something has gone wrong withthe test, as the control line should always develop, especially if thetest line appears.

Referring now to FIG. 3, which is a cross section of an apparatus forilluminating and capturing coupon images, in one preferred embodimentthe coupon reader 10 includes a light diffusing assembly 34 comprisingside walls 36 having reflective interior surfaces 38 and a negativeaxicon lens 40. The negative axicon lens 40 alters the path of lightrays 42 emitted from light source 48 and passing thought the lens suchthat the light rays 42 reflect off the interior surfaces 38 prior toreaching the coupon surface 44. The negative axicon lens 40 is designedto prevent any light from directly impinging on the coupon surface 44.All LED output light is first operated on by the light diffusingassembly 34 before it impinges on the coupon. An optimum shape for thelight diffusing assembly 34 is empirically determined, but it has beenfound that for illuminating a rectangular coupon, a concave structurethat has a rectangular cross-section works well if the lower skirt ofthe structure approximates the coupon's outer shape.

In one embodiment, the light diffusing assembly comprises side walls 36which form a rectangular cross-section approximately 8 cm on a side. Theside walls 36 meet the coupon surface 44 at angles that maximizeuniformity of illumination over the coupon surface and compensate forthe light source's off-center location. In a preferred embodiment, theincluded angle 46 between the side walls 36 and the coupon surface 44is:

Face 1=70 degrees

Face 2=70 degrees

Face 3=70 degrees

Face 4=70 degrees

In one embodiment, the distance between the coupon surface 44 and thenegative axicon lens 40 is 15 cm.

Diffuse reflection may be obtained from the reflective interior surfaces38 by covering them with a flat or gloss white paint, or by constructingthe structure using a white polymer. However, commercially availablewhite polymers may absorb a significant amount of the optical powerimpinging on them, and this may need to be taken into consideration.

Optionally, the reflective interior surfaces 38 may be covered with acolored coating rather than a white coating. A colored coating may bedesirable in circumstances where a specific light spectrum distributionis preferred for illuminating certain types of coupon. In a furtheroption, the reflective interior surfaces may be covered by a hybridcoating that provides a mixture of specular and diffuse reflection.

Axicons are special lenses that have at least one active surface that isconic in shape. They will typically take a point source of light andtransform it into either a line or circle of light. A negative axiconlens is used for coupon reader 10. Typical commercially available axiconlenses are designed to form a specific geometric shape, and for thatreason are considered “positive axicons.” The negative axicon, incontrast, takes a point of light and transforms it into an annular fanof light. Instead of a centrally located solid cone of material as in apositive axicon, the negative axicon has a conic pocket into whichsource light expands.

Refraction at the conic top surface and at the optionally planar lowersurface cause rays from a point source to be distributed over a range ofangles determined by the conic profile, the distance of the source fromthe lens, the lens thickness, and the refractive index of the lensmaterial. Angular emission of the lens can be tuned so that no lighthits the coupon surface 44 without first reflecting off the reflectiveinterior surfaces 38. That is, in a preferred embodiment, no lightpassing through the negative axicon lens 40 falls directly on the couponsurface 44, particularly at the spot immediately below the lens where ahotspot would normally occur. In this way, all light impinging on thecoupon surface 44 is diffused light re-emitted or reflected by thereflective interior surfaces 38 and the uniformity of illumination overthe coupon is greatly improved, even if the coupon is in close proximityto the light source and lens.

It is not necessary that the conic profile of the negative axicon lens40 be a simple cone. By adjusting the surface profile of the lens, thedistribution of emitted optical power can be modified in an angularsense. Since the objective of the negative axicon lens and lightdiffusing assembly is to provide diffused illumination, minor errors inthe surface profiles are not critical. In one embodiment, the lenses aremanufactured on a CNC lathe, followed by vapor polishing to removemacroscopic tooling marks.

In one embodiment, a negative axicon lens with planar output surface forsupplying optical power to the reflective interior surfaces isconstructed of clear polycarbonate. In one embodiment, the negativeaxicon lens has the following dimensions: the overall height of the lensmay be 3.5 mm, the diameter may be 10 mm, and the maximum depth of thecone may be 2.5 mm.

In one embodiment, the cone profile is described as:

Radius=1.98543z−0.18142z ²+0.04361z ³ (where z is the distance in mmfrom the cone's vertex).

In one embodiment, nonuniformity of illumination across the couponsurface is compensated for by color-based ratiometric analysis of pixeldata. Most color cameras provide at least three color sub-pixels percolor pixel; typically red, green, and blue sub-pixels. It has beenfound that light intensity variations over a coupon due to geometriclighting issues are very similar in the three color sub-pixels. Mostlateral flow immunoassay coupons use colloidal gold as the line colorantand its peak absorption is typically in the wavelength range of 540 nmto 580 nm, which is principally apparent in the camera pixels sensitiveto green and red. It has been discovered that by using the color pixelsto form “ratiometric” or, termed slightly differently, “color-ratio”pixels, that is a pixel having an intensity corresponding to the ratioof two sub-pixels, an unexpected and remarkable reduction in backgroundnonuniformity is seen. For the coupons having the common colloidal goldcolorant, the red and green sub-pixels are typically most useful in thisregard, but creating color-ratio pixels using other color sub-pixels maybe preferred for other colorants.

FIG. 4 shows raw intensity data for the green and red, column-summedsub-pixels of a coupon channel imaged using a solid state white lightflash illuminator. Also shown are the column-summed normalizedcolumn-summed green-to-red color-ratio pixel values, forming an“intensity profile.” This coupon had previously been exposed to a liquidcontaining the target substance, and hence displayed the target patternindicating a positive result. In this case, the target patternconstituted a line having both red and green color components runningthe width of the channel. The coupon was imaged and the image pixelswere divided into columns running parallel to the colorimetric lines.The values of the pixels in each column were summed to create the pixeldata sets shown. The large signal perturbation on the left side of FIG.4 exhibits the two absorbance maxima at approximate pixel locations 65and 185, respectively, due to colorimetric reactions on the test strip.The pixel signal minimum (with maximum divergence from the mean value)on the right, between pixel positions 400 and 500 is due to the controlline and should always be present if the coupon is working properly.Note that there are significant intensity fluctuations in the baselinesof both the red and green data sets.

In one embodiment, a new variable is created when the green pixelintensities are divided by the corresponding red pixel intensities, tocreate green-to-red color-ratio pixels and the resultant curve isnormalized to an arbitrary value of 1.0 (or 100%) at some arbitrarypixel location distant from the test and control lines. The resultingcolor-ratio pixel values are also shown in FIG. 4, normalized to a valueof 100. Note the removal of spurious non-signal fluctuations seen in thegreen and red pixel backgrounds in sections away from the threeabsorbance maxima, caused by variations in the background light level.This allows the test and control lines to show up much more clearly asdeviations from a near-constant color-ratio pixel background signal.

The fluid dynamic and chemical processes that create the test andcontrol lines are typically identical or very similar. For most coupons,even those with multiple identification channels, all test and controllines produce pixel signal responses that are similar in shape andsubstantively differ only in overall intensity. Therefore, the relativeconcentration of a targeted material may be estimated by simplycomparing the maximum deviation of the color-ratio pixel signal frombackground and comparing it to the maximum deviation of the fully-formedcolor-ratio pixel control line. However, this may not be the mostsuitable method for test lines that have a small peak intensity becausethere may be too high of a signal-to-noise ratio to determine the peakvalue accurately. In addition, the test and target lines are of finitewidth and vary in intensity over the line's width. Therefore, thequantity of target material in the sample is more appropriatelyproportional to the integrated total response over the entire linewidth. Using the integrated response over the entire line width willalso serve to smooth out pixel signal noise. For example, if a signal isaveraged over 100 pixels, random background noise as a fraction of themean signal value will typically be reduced by a factor of 10 timescompared to the noise associated with a single pixel measurementprocess.

In one embodiment, the following mathematical strategy and correspondingalgorithm automatically provide a measure of the test and control lineintegrated areas, and the degree of match between the shapes or“intensity profiles” of the two lines. This technique yields the pixel(or stated slightly differently, “coupon”) location of the test sectionintensity profile that best matches the reference pattern. That is, theset of contiguous pixels in the test section having an intensity profilethat most closely matches that of the reference pattern. The control andtest line locations may indicate whether the assay coupon was correctlyassembled or not, or if it was subsequently mishandled by the user.

The coupon typically includes a control line, which develops even in theabsence of the target substance to indicate that the coupon isfunctioning properly and that the correct procedures were used to exposeand develop the coupon. In one embodiment, the control line is used asthe source of reference information that permits the coupon reader todetermine whether a test line is present or absent. In otherembodiments, a different source of reference information may be used,such as a pre-printed line on the coupon, data from a different coupon,or an abstract representation of a developed control line, showing theexpected result when the tested for substance is present.

Referring to FIG. 5, because the test and control lines extend acrossthe width of each coupon channel, the color-ratio pixels are dividedinto columns running parallel to the test and control lines and thensummed across each column. Each column sum defined in this way isidentified by pixel position and referred to as a “pixel”, and the valueof the sum at that position is termed the “pixel value” for thatposition. The color-ratio pixel signal values associated with a coupon'stest and control lines can be approximated as the sum of a slowlyvarying background signal and a characteristically-shaped nonlinearabsorbance signature, Y*(i), where “i” is pixel position. The contiguousset of color-ratio pixels between two pixel-positions q and r (wherer−q=m), that encompass the control line, for example the 40 pixelsbetween pixel positions 430 and 470 in FIGS. 4 and 5, is such acharacteristic control line signature Y*(i). In one embodiment, atwo-variable nonlinear least squares regression technique may be used totest for the presence of signal or “test” lines with absorbancesignatures similar to Y*(i). This is done by comparing color-ratio pixeltest section data, Y(i), to a fitting equation, Y_(f)(i), which is givenas

Y _(f)(i)=a+b×i+c×Y*(i)

In this equation, “a” is the average baseline value, “b” is the rate ofbaseline change per pixel, and “c(p)” is a measure of thegoodness-of-fit of experimental data over a set of pixels of equal widthto the reference signature, Y*(i), and starting at pixel index “p”. Asnoted, in the example of FIG. 5, this set has about 40 scalar values,each representing a column sum of color-ratio pixels, that collectivelyform the control line. To obtain an unbiased measure of thisgoodness-of-fit, we minimize the sum of squares of the deviationsbetween the experimental data (that is, the intensity profile of eachset of contiguous m pixels in the test section) and the curve fit,Y_(f)(i),

η=Σ_(i=p) ^(p+m) [Y(i)−Y _(f)(i−p)]²

Where p=the starting pixel position and m (as noted above) is the numberof values in the Y*(i) set of values and i=0, is the starting pixelposition of Yf(i). The values of a(p), b(p) and c(p) yielding theminimum value of η are found (in the manner described below) repeatedly,for each value of “p” over the test section of the coupon. In theexample of FIG. 4, the values of c(p) are shown for every value of p,from p=1 to p=600, but this is done to show that the least squares fitof Y*(i) with itself yields a c(p) of 1. To determine whether a targetsubstance has been found, it would be more typical to find the values ofc(p) just in the test section of the coupon, which would be for p from 1to 350, in the example of FIG. 5. The maximum value of c(p) or“c(p)_(max)” is compared to a threshold, and if it exceeds thethreshold, a detection is declared.

Skilled persons will recognize that solving for the c(p) that minimizesn, for each value of p, and then thresholding the largest c(p), is morelikely to yield a detection in the instance in which a section of thecoupon not only has some intensity (that is, contrast or differencerelative to a background noise level, which may be shown as darknessagainst a white background), but also where the portion tested has asimilar shape of intensity profile to Y*(i). As shown in FIG. 4, whenthe value of p results in the control line being placed directly overitself, c(p) attains its maximum value of “1.”

Also, the value of c(p)_(max) is not directly affected by position of p,within the pixel linear array, so that c(p)_(max) may occur at someother location than the value of p that would be expected based oninformation from the coupon manufacturer. This has a benefit, becausecoupons sometimes have flaws in the position of the prospective testline, and it is accordingly advantageous to “look” for a test line in awider area than only where it would be expected based on the intendedposition. Moreover, when c(p)_(max) is found, the pixel position “p” mayyield valuable information concerning the state of the coupon that hasbeen tested.

The procedure determining the values of the parameters “a(p)”, “b(p)”and “c(p)” to minimize η are well-known in statistical mathematics. Theprocedure is performed over the test pixel section to find a value of“c(p)” for each possible pixel starting position “p”. When thisprocedure is performed, it is found that the best-fit value of “c” isgiven by the following equation:

C=K ₁ ×S _(iY) +K ₂ ×S _(Y) −K ₃ ×S _(YS)

The values K₁, K₂, and K₃ are constants that are derived based on thereference signature Y*(i), and are easily calculated by someoneknowledgeable in statistical mathematics. The other equation factors arecalculated for each sectional pixel array of Y(p) through Y(p+m) to beexamined as follows:

S _(iY)=Σ_(i=p) ^(p+m) i×Y(i) . . . S _(Y)=Σ_(i=p) ^(p+m) Y(i) . . . S_(YS)=Σ_(i=p) ^(p+m) Y(i)×Y*(i−p)

The three summations are easily and quickly calculated and the constantsK₁, K₂, and K₃ are only calculated once: After that they may be storedas look-up values for the channel. If the pixel data presented forcomparison is the reference pixel array Y*_(i) itself, the value of“c(p)” will be exactly 1.0 as discussed previously.

As previously discussed, the test line, or pattern, in most cases isessentially identical in shape (that is, in cross-sectional variation inintensity) to the control line, but less intense overall. The value of“c” will have a maximum value when the signal data most closely matchesthe control line shape Y*(i). This allows identifying the location ofeach test line center point while the value of “c” represents therelative integral size of the discovered test line as compared to thecontrol line.

This method is favored for several reasons:

-   -   Small variations in the baseline are common due to lighting        variations, shadows, and residual color in the coupon substrate.        This method automatically estimates and corrects for a        non-constant baseline. The value of c(p) is an unbiased best        estimate of the baseline-corrected integral value.    -   Due to the use of all data points associated with a line,        signal-to-noise is improved and a wide dynamic range is realized        of as much as 100 to 1. That is, a test line with a value of        c(p)_(max) on the order of 0.01 may be detected.    -   The value of c(p)_(max) reflects the target substance        concentration, which allows development of a calibration curve        that quantitatively correlates the targeted material's        concentration with the value of c(p)_(max).    -   The method allows a quantitative determination that a test        line's response is statistically above background noise.    -   The method provides the lines' peak value location, which is        useful in identification of manufacturing or user errors.    -   While the method can be applied to raw data as well as        ratiometric data, the integral value from ratiometric data        provides a measure of signal strength that is independent of        absolute light intensity or light exposure time as long as the        color channels all change proportionately the same when these        factors are varied. This is a reasonable assumption for changes        in exposure time or temperature, since both the control line and        test line will see the same environmental shifts.

In a preferred embodiment, for a multichannel coupon the three constantsK1, K2, and K3 are empirically determined for each individual channel byrunning one or more assays and calculating these least-squares constantsfor each channel's control line. This may be of particular value ifthere are differences in channel construction or if assay reagents aresignificantly different among the channels.

In addition, in a multi-channel coupon the channel control lines may becompared one against another and these relative “c” values stored in thedevice's memory. During later use, the control lines can be compared toeach other to see if any have deviated from the expected relative “c”values. The device may then issue a warning to the user that there maybe a problem with one or more channels.

In a preferred embodiment, digital images of the coupon are repeatedlytaken over a period of time, starting shortly after addition of thesample and well before the time period suggested by the manufacturer haspassed. These images are processed sequentially to determine if a testline has developed. As soon as detection positive result is found, ahuman perceivable signal is given. Depending on the coupon type, it ispossible to gather useful information as early as 1 minute afterdevelopment has been initiated. Some explosives coupons have a peakcolor at 2 minutes after application of a test fluid, and noticeablyfade after 3 minutes. Accordingly, the time after exposure at which afirst digital image is formed may be as little as 10 seconds or even 1second. The techniques described herein have been shown to reduce thetime to detection for bio-coupons to as little as 5 minutes, a third ofthe time period that the manufacturer recommends waiting, beforeevaluating an exposed coupon.

Accordingly, in embodiments the taking of digital imagery begins wellbefore the manufacturer's development time has passed. In variousembodiments, the taking of digital imagery begins at least 4, 3, 2 and 1minute prior to the passing of the recommended time to develop, afterexposure to said test fluid mixture. Described in different terms, invarious embodiments a first digital image is formed no more than 1, 2, 3and 4 minutes after the coupon channel is exposed to said test fluidmixture.

In a further inventive feature, digital imagery is also repeatedly takenof the control section of each channel, beginning shortly after exposureto a test fluid mixture. This permits early detection of a coupon thathas been ineffectively exposed to the test fluid mixture. As soon asineffective exposure is determined, the test can be terminated and a newtest begun, thereby greatly decreasing the time before an accuratereading is determined.

In a further embodiment, the sequence of images are integrated together,to enhance the signal to noise ratio and, in some instances, provideearlier detection than would otherwise be possible. In one variant, timeintegration is performed on the raw pixels, but in another least squaresfitting as described above is applied to the data from each digitalimage and the multiple results are then integrated over time.

The invention may be embodied in other specific forms besides and beyondthose described herein. The foregoing embodiments are therefore to beconsidered in all respects illustrative rather than limiting, and thescope of the invention is defined and limited only by the appendedclaims and their equivalents, rather than by the foregoing description.

1. A method of reading a coupon channel that displays a test pattern ina test section after being exposed to a target substance, said methodincludes providing a device having a computer readable memory, a digitalcamera, a logic assembly and a user interface; providing a color-ratiopixel target pattern representation, representative of an exposed and atleast partially developed coupon channel control section pattern;placing said color-ratio pixel target pattern into said memory; placingsaid coupon in said device and exposing said coupon channel to a testfluid mixture; automatically using said digital camera to take a digitalimage of said coupon channel test section after said exposure, saiddigital image formed of color pixels, each containing at least twosub-pixels representing the intensity of two different colors; and a.wherein the improvement in said method includes for each color pixelforming a color-ratio pixel by forming the ratio of a first one of saidtwo subpixels with the other, and automatically using said logicassembly to compare said color-ratio pixels in said test section asshown in said digital image to said color-ratio pixel target patternrepresentation to determine if said test section passes a similaritythreshold; and in response to a test section passing said similaritythreshold, automatically providing a human perceptible indication thatthe target substance has been detected.
 2. The method of claim 1,wherein said coupon channel includes a control section that develops atarget pattern after exposure to said test fluid mixture, without regardto the presence of said target substance in said test fluid mixture, andwherein said color-ratio pixel target pattern representation is providedby digital imagery from said digital image of said target pattern insaid control section, and further processing in which said color-ratiopixel values are computed and wherein said providing a color-ratio pixeltarget pattern representation occurs after said exposing said couponchannel to a test fluid mixture.
 3. The method of claim 1, wherein saidcolor-ratio pixel target pattern representation is provided by taking acolor digital photograph, having color pixels, of a coupon channel thathas been exposed to said target substance after a predetermineddevelopment time has elapsed since said exposure and computing saidcolor-ratio pixels from said color pixels.
 4. The method of claim 1,wherein said threshold is user adjustable and further including theinitial step of adjusting said threshold.
 5. The method of claim 1,wherein said coupon channel is hosted on a coupon having a singlechannel only.
 6. The method of claim 1, wherein said coupon channel ishosted on a coupon having multiple channels.
 7. A method of reading acoupon channel that displays a test section pattern after being exposedto a target substance, said method includes providing a device having acomputer readable memory, a digital camera, a logic assembly and a userinterface; providing a pixel target intensity profile representation,representative of an exposed and at least partially developed couponchannel control section intensity profile; placing said pixel targetintensity profile into said memory; placing said coupon in said deviceand exposing said coupon channel to a test fluid mixture; automaticallyusing said digital camera to take a digital image of said coupon channeltest section after said exposure; and a) wherein the improvement in saidmethod includes finding the contiguous set of pixels from said testsection of said coupon channel that best matches the intensity profileof said target pattern representation and determining if this best matchset of pixels exceeds a similarity threshold and, in response to a bestmatch set of pixels passing said similarity threshold, automaticallyproviding a human perceptible indication that the target substance hasbeen detected.
 8. The method of claim 7, wherein said coupon channel ishosted on a coupon having a single channel only.
 9. The method of claim7, wherein said coupon channel is hosted on a coupon having multiplechannels.
 10. The method of claim 7, wherein said contiguous set ofpixels that best matches the intensity profile of said target patternrepresentation is the set of contiguous pixels that has the best leastsquares match.
 11. The method of claim 7, wherein linear bias issubtracted out of each contiguous set of pixels to obtain the bestmatch.